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Spices are susceptible to mycotoxin contamination which can cause gastrointestinal and adverse central nervous symptoms in humans, which highlights the importance of assessing the risk of their consumption on a daily basis. The aim of this study was to assess the risk of mycotoxin intake from spices in routinely prepared Lebanese dishes. 150 households were interviewed about their usage of 27 type of spices and 6 routinely prepared Lebanese dishes. Results showed a high variability in consumption levels. Among the investigated dishes, the minimum number of spices that were consumed in a dish was 13 while the maximum was 18. The mean intake of one spice ranged from 0.26 g/portion observed for cloves to 5.37 g/portion for cinnamon, with its intake per portion more than 1 g in 2/3 of dishes. 20% of portion sizes of coriander, cinnamon and fennel, had an intake exceeding 5 g/portion. Ochratoxin A (OTA) Probable Daily Intake (PDI) had a mean of 0.11 ng/kg-bw/day. Mean PDI of fumonisin B1 (FB1) was 79.3 ng/kg-bw/day. Aflatoxin B1 (AFB1) PDI had a mean of 1.55 ng/kg-bw/day. The Margin of Exposure (MOE) of AFB1 ranged from 108.10 to 4444.44. The present study showed that the risk of AFB1 from spices is a matter of concern while the risk of OTA and FB1 is limited with the exception of FB1 from garlic and onion.

Aflatoxins are fungal metabolites extensively produced by many different fungal species that may contaminate a wide range of agricultural food products. They have been studied extensively because of being associated with various chronic and acute diseases, especially immunosuppression and cancer, and their presence in food is strictly monitored and regulated worldwide. Aflatoxin detection and measurement relies mainly on chemical methods usually based on chromatography approaches, and recently developed immunochemical based assays that have advantages but also limitations, since these are expensive and destructive techniques. Nondestructive, optical approaches are recently being developed to assess presence of contamination in a cost and time effective way, maintaining acceptable accuracy and reproducibility. In this paper are presented the results obtained with a simple portable device for nondestructive detection of aflatoxins in almonds. The presented approach is based on the analysis of fluorescence spectra of slurried almonds under 375 nm wavelength excitation. Experiments were conducted with almonds contaminated in the range of 2.7–320.2 ng/g total aflatoxins B (AFB1 + AFB2) as determined by High Performance Liquid Chromatography with Fluorescence Detection (HPLC/FLD). After applying pre-processing steps, spectral analysis was carried out using a binary classification model based on Support Vector Machine (SVM) algorithm. A majority vote procedure was then performed on the classification results. In this way we could achieve, as best result, a classification accuracy of 94% (and false negative rate 5%) with a threshold set at 6.4 ng/g. These results illustrate the feasibility of such approach in the great challenge of aflatoxin detection for food and feed safety.

Forty-five samples of a landrace of sweet pepper (Capsicum annuum) widely cultivated in Basilicata (Italy) were screened for 17 mycotoxins and potential toxigenic fungal species. Two different LC-MS/MS methods were used for the determination of aflatoxins, ochratoxin A (OTA), Fusarium mycotoxins zearalenone (ZEA), fumonisins (FB1 and FB2), nivalenol (NIV), deoxynivalenol (DON), T-2 and HT-2 toxins and Alternaria mycotoxins altenuene (ALT), alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TTX) and tenuazonic acid (TeA). Frequency of potential toxigenic fungal species occurrence was: 87% Aspergillus Sect. Nigri; 58% Aspergillus Sect. Flavi; 38% Aspergillus Sect. Circumdati; 42% Alternaria spp.; 33% Penicillium spp. and 20% Fusarium spp. Frequency of mycotoxin occurrence and mean of positives were: 51% OTA, 29.5 µg/kg, 5 samples above the EU limit of 20 µg/kg; 31% aflatoxins, 12.8 µg/kg, two samples above the EU limit of 5 µg/kg for aflatoxin B1; 91% ZEA, 1.4 µg/kg; 78% FB2, 7.6 µg/kg; 58% FB1, 22.8 µg/kg; 38% NIV, 39.5 µg/kg; 36% DON, 6.9 µg/kg; 20% T-2 toxin, 5.6 µg/kg and 22% HT-2 toxin, 13.8 µg/kg. For the Alternaria mycotoxins, 100% of samples contained TeA, 4817.9 µg/kg; 93% TTX, 29.7 µg/kg; 56% AOH, 114.4 µg/kg; 33% AME, 13.0 µg/kg and 9% ALT, 61.7 µg/kg. Co-occurrence of mycotoxins in each sample ranged from 2 to 16 mycotoxins (mean 7). No statistical correlation was found between moulds and their mycotoxins occurrence. Within the four groups of peppers collected herein (fresh, dried, grounded and fried) higher percentages of contamination and mycotoxin levels were measured in grounded peppers, whereas much lower values were observed for fried peppers. The high percentages of positive samples and the high levels of some mycotoxins observed in this study confirm the susceptibility of peppers to mycotoxin contamination and claims for an improvement of the conditions used during production and drying process.

Metabolic profile of urine from piglets administered with single boluses contaminated with mycotoxin mixture (deoxynivalenol, aflatoxin B1, fumonisin B1, zearalenone, and ochratoxin A) were studied by 1H NMR spectroscopy and chemometrics (PCA, PLS-DA, and OPLS-DA). The mycotoxin levels were close to the established maximum and guidance levels for animal feed (2003/100/EC and 2006/576/EC). Urine samples were obtained from four groups of four piglets before (control, C) or within 24 h (treated, T) after receiving a contaminated boluses with increasing doses of mycotoxins (boluses 1–4). For the two highest dose groups, the urines were collected also after one week of wash out (W). For the two lowest doses groups no significant differences between the C and T samples were observed. By contrast, for the two highest doses groups the T urines separated from the controls for a higher relative content of creatinine, p-cresol glucuronide and phenyl acetyl glycine and lower concentration of betaine and TMAO. Interestingly, a similar profile was found for both W and T urines suggesting, at least for the highest doses used, serious alteration after a single bolus of mycotoxin mixture., Highlights • A single dose of mycotoxins mixture (DON, AFB1, FB1, ZEN, and OTA ) could produce serious impairments of gut microbiota of piglets. • The explorative metabolomic analysis on urine piglets, fed with boluses contaminated with mycotoxins mixtures, was performed. • NMR-based MVA resulted in a higher urinary concentrations of creatinine, p-cresol glucuronide and phenyl acetyl glycine and lower concentrations of betaine and TMAO after treatment with respect to own controls.

The determination of mycotoxin and metabolite concentrations in human and animal urine is currently used for risk assessment and mycotoxin intake measurement. In this study, pig urine (n = 195) was collected at slaughterhouses in 2012 by the Swedish National Food Agency in three counties representing East, South and West regions of Sweden. Urinary concentrations of four mycotoxins, (deoxynivalenol (DON), zearalenone (ZEA), fumonisin B1 (FB1), and ochratoxin A (OTA)), and four key metabolites, (deepoxy-deoxynivalenol (DOM-1), aflatoxin M1 (AFM1, biomarker of AFB1), &alpha, zearalenol (&alpha, ZOL), and &beta, zearalenol (&beta, ZOL)) were identified and measured by UPLC-MS/MS. Statistically significant regional differences were detected for both total DON (DON + DOM-1) and total ZEA (ZEA + &alpha, ZOL + &beta, ZOL) concentrations in pig urine from the three regions. These regional differences were in good agreement with the occurrence of Fusarium graminearum mycotoxins (DON + ZEA) in cereal grains harvested in 2011 in Sweden. There were no statistically significant differences in FB1, AFM1 and OTA urinary concentrations in pigs from the three regions. The overall incidence of positive samples was high for total ZEA (99&ndash, 100%), total DON (96&ndash, 100%) and OTA (85&ndash, 95%), medium for FB1 (30&ndash, 61%) and low for AFM1 (0&ndash, 13%) in the three regions. Urinary mycotoxin biomarker concentrations were used to estimate mycotoxin intake and the level of mycotoxins in feeds consumed by the monitored pigs. The back-calculated levels of mycotoxins in feeds were low with the exception of seven samples that were higher the European limits.

The emerging Alternaria mycotoxins tenuazonic acid (TeA), alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) and tentoxin (TTX) are not yet regulated worldwide awaiting for more data on their toxicity and natural occurrence. A total of 131 samples of spices (94) and herbs (37) were collected in Beirut (Lebanon) markets and analysed for TeA, AOH, AME, ALT and TTX by using a UPLC-MS/MS method based on ‘extract, dilute and shoot’ approach. High percentage (89%) of samples were contaminated by 1–5 mycotoxins whereas 14% of samples were contaminated with a mixture of TeA, AOH, AME and TTX. TeA was the predominant mycotoxin with the highest percentages of positive samples (76%), followed by AME (46%), TTX (37%), AOH (34%) and ALT (5%) with overall mean/max levels of 2453.2/106792.8, 17.1/306, 14.8/179.4, 35.0/636.4 and 2.8/22.1 μg/kg, respectively (middle bound approach). Higher percentage of positive samples and mean mycotoxin levels were observed in spices as compared to herbs. The spices with higher levels of mycotoxin contamination were, in descending order, red chili, paprika, caraway, coriander seeds, onion powder, white pepper and garlic powder whereas within the herbs they were corn silk, violets and marjoram. Based on the mean levels of TeA measured in different spices and herbs, the daily consumption of >3.3 g red chili, >3.7 g paprika, >16.7 g caraway or >36 g of corn silk will exceed the threshold of toxicological concern (TTC) of TeA (1,500 ng/kg bw per day). For AOH or AME the TTC (2.5 ng/kg bw/day) will be exceeded by a daily consumption >0.5 g of white pepper, >0.9 g of onion powder, > 1.4 g of garlic powder, >1.7 g of black pepper, >1.9 g of sage, >3.3 g of violets or > 4.4 g of thyme leaves. This study provide large information on the natural occurrence of Alternaria mycotoxins in spices and herbs useful for the human exposure assessment and future regulation.

Twelve different approaches commonly used for the simultaneous LC tandem MS (MS/MS) determination of mycotoxins (deoxynivalenol, aflatoxins, ochratoxin A, T-2 and HT-2 toxins, fumonisins, and zearalenone) were tested in cereals and feed materials. They comprised different extraction solvents, types of cleanup [solid-phase extraction, QuEChERS, and immunoaffinity (IMA)], and calibration approaches (external or matrix-matched). The percentage of mycotoxins with acceptable recovery, according to Regulation (EC) No. 401/2006, ranged from 9 to 100%. The approach giving the highest percentage of acceptable results was selected and further tested for corn, rice, and feed spiked at three different mycotoxin levels (low, medium, and high). The method is based on extraction with MeOH–water (70 + 30, v/v) and cleanup with two multiantibody IMA columns. For corn and rice spiked at low mycotoxin levels, a significant matrix effect was observed and was compensated by using 13C calibration. At higher mycotoxin levels (medium and high), matrix effects were negligible as no significant differences were observed for the majority of recovery results calculated by 13C calibration and external calibration. Although the proposed method still needs improvement in terms of accuracy and, to a lesser extent, precision, it was successfully tested with four proficiency tests in buckwheat, corn, rice, and feed, giving acceptable z-scores for 97% (34 out of 35) of results.

Deoxynivalenol (DON) is an important mycotoxin produced by several species of Fusarium. It occurs often in wheat grain and is frequently associated with significant levels of its modified form DON-3-glucoside (DON-3-Glc). Ozone (O3) is a powerful disinfectant and oxidant, classified as GRAS (Generally Recognised As Safe), that reacts easily with specific compounds including the mycotoxins aflatoxins, ochratoxin A, trichothecenes and zearalenone. It degrades DON in aqueous solution and can be effective for decontamination of grain. This study reports the efficacy of gaseous ozone treatments in reducing DON, DON-3-Glc, bacteria, fungi and yeasts in naturally contaminated durum wheat. A prototype was used to dispense ozone continuously and homogeneously at different concentrations and exposure time, in 2kg aliquots of durum wheat. The optimal conditions, which do not affect chemical and rheological parameters of durum wheat, semolina and pasta, were identified (55g O3h-1 for 6h). The measured mean reductions of DON and DON-3-Glc in ozonated wheat were 29% and 44%, respectively. Ozonation also produced a significant (p

Deoxynivalenol (DON) exposure is estimated by the combined measures of urinary DON and DON-glucuronides. In this study, data from single-mycotoxin (SM) and a multimycotoxin (MM) methods were compared for 256 Swedish adult urine samples. Both methods included ?-glucuronidase predigestion, immunoaffinity enrichment, and LC-MS/MS. However, the specific reagents, apparatus, and conditions were not identical in part because the MM method measures additional mycotoxins. DON was detected in 88 and 63% of samples using the SM and MM methods, respectively, with the following mean and median concentrations: SM, mean = 5.0 ng/mL, SD = 7.4, range of positives = 0.5-60.2 ng/mL, median = 2.5 ng/mL, IQR = 1.0-5.5 ng/mL; MM, mean = 4.4 ng/mL, SD = 12.9, range of positives = 0.5-135.2 ng/mL, median = 0.8 ng/mL, IQR = 0.3-3.5. Linear regression showed a significant, albeit modest, correlation between the two measures (p = 0.0001, r = 0.591). The differences observed may reflect subtle handling differences in DON extraction and quantitation between the methods.

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxin LC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancer region, Transkei, South Africa. Fifty-three female participants donated part of their maize-based evening meal and first void morning urine, which was analysed both with sample clean-up (single and multi-biomarker) and by a 'dilute-and-shoot' multi-biomarker method. Results were corrected for recovery with LOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean ± standard deviation 0.342 ± 0.466 ng/mg creatinine) and deoxynivalenol (100%; mean 20.4 ± 49.4 ng/mg creatinine) after hydrolysis with b-glucuronidase. The multi-biomarker 'dilute-and-shoot' method indicated deoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with b-glucuronidase and immunoaffinity clean-up determined zearalenone (100%; 0.529 ± 1.60 ng/mg creatinine), FB1 (96%; 1.52 ± 2.17 ng/mg creatinine), a-zearalenol (92%; 0.614 ± 1.91 ng/mg creatinine), deoxynivalenol (87%; 11.3 ± 27.1 ng/mg creatinine), b-zearalenol (75%; 0.702 ± 2.95 ng/mg creatinine) and ochratoxin A (98%; 0.041 ± 0.086 ng/mg creatinine). These demonstrate the value of multi-biomarker methods in measuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinary deoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei.

The performances of four LC-MS/MS methodologies for determination of up to eight mycotoxin biomarkers in human urines were compared by involving three laboratories that analysed common urine samples spiked at two levels of each biomarker. Each laboratory received a calibration solution, spiked urines and the corresponding unspiked urine. The two spiking levels for each biomarker were chosen by considering the levels naturally occurring in human urines and the limits of quantification of the LC-MS/MS methodologies used by the participating laboratories. The results of each laboratory were evaluated for their z-score values. The percentage of satisfactory z-scores (| z | < 2) were: 100% for deoxynivalenol, de-epoxy deoxynivalenol, aflatoxin M1, β-zearalenol and zearalenone, 87% for α-zearalenol, 50% for ochratoxin A and 42% for fumonisin B1. Good method performances were obtained for most biomarkers at the levels tested in this study, as demonstrated by the overall percentage of satisfactory z-scores for all analytes (87%). Unsatisfactory/questionable z-scores (| z | ≯2) were obtained for fumonisin B1 (7/12 results), ochratoxin A (4/8 results) and ?-zearalenol (1/8 results). The percentage of satisfactory z-scores for fumonisin B1 and ochratoxin A increased from 42 to 83% for fumonisin B1 and from 50 to 62% for ochratoxin A when laboratories 1 and 2 used own calibrants. Factors that could explain the different results obtained for fumonisin B1 and ochratoxin A with provided and own calibration solutions could not be identified in this study and should be carefully investigated in future studies.

Maize harvested in the Centane region of the former Transkei, Eastern Cape Province, South Africa, by subsistence farmers has been shown over many seasons to be contaminated with fumonisin mycotoxins. However, there are limited data on the presence of other mycotoxins. Two multimycotoxin LC-MS/MS methods were applied to good and moldy maize samples, as separated by the farmers themselves from the 2011 harvest. One method involved extract cleanup on multitoxin immunoaffinity columns before LC-MS/MS analysis for aflatoxins, fumonisins, deoxynivalenol (DON), zearalenone (ZEN), and T-2 and HT-2 toxins. The other method was based on a "dilute-and-shoot" approach for the above mycotoxins and a wide range of other fungal secondary metabolites. Both methods showed high incidences of fumonisins B1 and B2 (FB1 and FB2) in good maize (100% for both by the first method, means were 2083 and 927 μg/kg for the two analogues; 93% for both by the second method, positive means of 2764 and 1050 μg/kg, respectively). All samples of moldy maize were contaminated (mean FB1 of 27.64 and 35.98 mg/kg, respectively; mean FB2 of 10.58 and 14.14 mg/kg, respectively). Comparison of the two methods for FB1 and FB2 over the entire range of samples gave R(2) values 0.9144 and 0.8859, respectively. Low levels of DON were found by both methods (positive means of 12 and 4.7 μg/kg in good maize, respectively, and of 14 and 5.8 μg/kg in moldy maize, respectively). ZEN was determined with positive means of 108 and 25 μg/kg in good maize, respectively, and of 111 and 135 μg/kg in moldy maize, respectively. No aflatoxins, OTA, or T-2 or HT-2 toxins were detected. A wide range of other Fusarium , Aspergillus , Alternaria , and Penicillium mycotoxins and secondary metabolites were determined.

The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels was assessed. Naturally-contaminated kernels were submitted to an electronic optical sorter or blanched, peeled, and manually sorted to visually identify and sort discolored kernels (dark and spotted) from healthy ones. The samples obtained from the two sorting approaches were ground, homogenized, and analysed by HPLC-FLD for their aflatoxin content. A mass balance approach was used to measure the distribution of aflatoxins in the collected fractions. Aflatoxin B1 and B2 were identified and quantitated in all collected fractions at levels ranging from 1.7 to 22,451.5 µg/kg of AFB1 + AFB2, whereas AFG1 and AFG2 were not detected. Excellent results were obtained by manual sorting of peeled kernels since the removal of discolored kernels (2.6%–19.9% of total peeled kernels) removed 97.3%–99.5% of total aflatoxins. The combination of peeling and visual/manual separation of discolored kernels is a feasible strategy to remove 97%–99% of aflatoxins accumulated in naturally-contaminated samples. Electronic optical sorter gave highly variable results since the amount of AFB1 + AFB2 measured in rejected fractions (15%–18% of total kernels) ranged from 13% to 59% of total aflatoxins. An improved immunoaffinity-based HPLC-FLD method having low limits of detection for the four aflatoxins (0.01–0.05 µg/kg) was developed and used to monitor the occurrence of aflatoxins in 47 commercial products containing apricot kernels and/or almonds commercialized in Italy. Low aflatoxin levels were found in 38% of the tested samples and ranged from 0.06 to 1.50 μg/kg for AFB1 and from 0.06 to 1.79 μg/kg for total aflatoxins.

Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M(1) (AFM(1)), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alpha-zearalenol (α-ZOL)/beta-zearalenol (β-ZOL), and fumonisin B(1) (FB(1)) can be used as urinary biomarkers. A liquid chromatographic-tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed-phase chromatography using a multi-step linear methanol-water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC-ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03-12 ng mL(-1), using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3-20%. Enzymatic digestion with β-glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time.